Hetero-assembling of spherical building blocks with well-defined spatial distribution holds great significance in developing chiral nanostructures. Herein, a strategy for hetero-assembling of gold nanoparticles(Au NPs) was demonstrated using rigid bifacial DNA origami as templates. By tuning the sizes and the fixed location of Au NPs on DNA origami, right-handed and left-handed Au NPs nanostructures were respectively constructed. Gel electrophoresis indicated the formation of the DNA origami-Au NPs complex and transmission electron microscopy(TEM) visually displayed the arrangement of Au NPs in these two chiral structures. The spatial configuration and 3D geometry of Au NPs were further illustrated by the stereographic TEM with tilting angles from ?30° to 30°. This strategy provides a universal approach to construct the asymmetrical 3D geometries, which may have potential applications in biomimicking and nanophotonics. Hetero-assembling of spherical building blocks with well-defined spatial distribution holds great significance in developing chiral nanostructures. Herein, a strategy for hetero-assembling of gold nanoparticles (Au NPs) was demonstrated using rigid bifacial DNA origami as templates. By tuning the sizes and the fixed location of Au NPs on DNA origami, right-handed and left-handed Au NPs nanostructures were respectively constructed. Gel electrophoresis indicated the formation of the DNA origami-Au NPs complex and transmission electron microscopy (TEM) shows displayed the arrangement of Au NPs in these two chiral structures. The spatial configuration and 3D geometry of Au NPs were further illustrated by the stereographic TEM with tilting angles from ~ 30 ° to 30 °. This strategy provides a universal approach to construct the asymmetrical 3D geometries, which may have potential applications in biomimicking and nanophotonics.